Molecular Structure of Human Chromosomes

337 Pages·1977·10.51 MB·English

by  Jorge Yunis (Eds.)

CHROMOSOMES IN BIOLOGY AND MEDICINE
Edited by
JORGE J. YUN1S, M.D.
New Chromosomal Syndromes, 1977
Molecular Structure oj Human Chromosomes, 1977
Molecular Structure
of Human Chromosomes
Edited by
JORGE J. YUNIS, M.D.
Medical Genetics Division
Department of Laboratory Medicine and Pathology
University of Minnesota, Medical School
Minneapolis, Minnesota
ACADEMIC PRESS New York San Francisco London 1977
A Subsidiary of Harcourt Brace Jovanovich, Publishers
COPYRIGHT €> 1977, BY ACADEMIC PRESS, INC.
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Library of Congress Cataloging in Publication Data
Main entry under title ·
Molecular structure of human chromosomes.
(Chromosomes in biology and medicine series)
Bibliography: p.
Includes index.
1. Human chromosomes. 2. Molecular genetics.
1. Yunis, Jorge J. II. Series: Chromosomes in
biology and medicine. [DNLM: 1. Cytogenetics.
2. Chromosomes—Ultrastructure. 3. Molecular
biology. QH600 M718]
QH600.M64 599'.9'θ48732 76-50672
ISBN 0-12-775168-8
PRINTED IN THE UNITED STATES OF AMERICA
List of Contributors
Numbers in parentheses indicate the pages on which authors' contributions begin.
G. F. BAHR (143), Department of Cellular Pathology, Armed Forces Institute
of Pathology, Washington, D. C.
GIORGIO BERNARDI (35), Laboratoire de Génétique Moléculaire, Institut de
Biologie Moléculaire, Université de Paris, Paris, France
RICHARD P. CREAGAN* (89), Department of Biology, Yale University, New
Haven, Connecticut
BERNARD DUTRILLAUX (233), Institut de Progénèse, l'Ecole de Médecine,
Paris, France
K. W. JONES (295), Institute of Animal Genetics, Edinburgh, Scotland
GABRIEL MACAYA (35), Laboratoire de Génétique Moléculaire, Institut de
Biologie Moléculaire, Université de Paris, Paris, France
P. L. PEARSON (267), Instituut voor Anthropogenetica, Leiden, Holland
POTU Ν. RAO (205), Department of Developmental Therapeutics, The Uni-
versity of Texas System Cancer Center, M. D. Anderson Hospital and
Tumor Institute, Houston, Texas
FRANK H. RUDDLE (89), Department of Biology, Yale University, New
Haven, Connecticut
DALE M. STEFFENSEN (59), Department of Genetics and Development,
University of Illinois, Urbana, Illinois
JEAN-PAUL THIERYt (35), Laboratoire de Génétique Moléculaire, Institut de
Biologie Moléculaire, Université de Paris, Paris, France
MICHAEL Y. TSAI (1), Medical Genetics Division, Department of Laboratory
Medicine and Pathology, University of Minnesota Medical School, Minnea-
polis, Minnesota
*Present address: Department of Physiology, School of Medicine, University of Con-
necticut Health Center, Larmington, Connecticut
I Present address: The Rockefeller University, New York, New York.
ix
χ List of Contributors
ANN M. WILLEY (1), Medical Genetics Division, Department of Laboratory
Medicine and Pathology, University of Minnesota Medical School, Minnea-
polis, Minnesota
JORGE J. YUNIS (1), Medical Genetics Division, Department of Laboratory
Medicine and Pathology, University of Minnesota Medical School, Minnea-
polis, Minnesota
Preface
In recent years, a number of important developments have led to a dramatic
increase in our ! understanding of chromosomes in biology and medicine. The
sequence arrangement, transcriptional capacity, and functional significance of
repeated and unique DNA sequences have been extensively studied. Refined
investigations of chromosome and chromatin structure have been made possible
with improvements in electron microscopic techniques, the use of premature
chromosome condensation, and in situ hybridization. By the use of various dyes
and treatments, mitotic chromosomes can now be visualized as having character-
istic banding patterns, facilitating identification of individual chromosomes.
Relationships between the human karyotype and those of other primates have
been determined using differential staining techniques and comparative analysis
of repeated DNA sequences. Gene mapping has been greatly accelerated with the
use of rodent - human somatic hybrids.
Reviews of advances on the organization of the eukaryotic genome have been
available for some time, yet they have not focused on the genome of greatest
interest to us, that of man. The aim of this volume is to fill this gap by bringing
together authoritative contributions encompassing much of the knowledge
available on the fine structure and molecular organization of the human genome.
Geneticists, molecular biologists, and cytogeneticists, particularly those in-
terested in the human genome, will find this book to be an invaluable guide.
Jorge J. Yunis
xi
1
Molecular Organization and
Function of the Human
Genome
JORGE J. YUNIS, MICHAEL Y. TSAI,
and ANN M. WILLE Y
I. Introduction 1
II. Renaturation Studies of Eukaryotic DNA 2
III. Gene Number and Genome Transcription 3
IV. Correlation of Transcription with Sequence Arrangement
Patterns 6
V. Fractionation of Native Double-Stranded DNA 6
VI. Hybridization of Nuclear RNA's to DNA in CsCl Density
Gradients 13
VII. Chromosomal Localization of Specific DNA and RNA
Components 15
VIII. Fine Structure of Chromosomes 20
IX. Conclusion 25
References 27
I. INTRODUCTION
A major obstacle encountered in studying the genome of higher eukaryotes
including man has centered around the large amount of DNA present in the
nuclei of these cells. Compared to the extensively studied prokaryotic genome of
Escherichia coli, consisting of 3.2 X 106 nucleotide pairs (Cairns, 1963), the
genome of the lower eukaryote Drosophila melanogaster contains approximately
1
2 Jorge Yunis, Michael Tsai, and Ann Willey
50 times that amount of DNA (Laird, 1971); while the human genome contains
1000 times the amount of DNA of E. coli, or 2.7 X 10 9 nucleotide pairs (Sober,
1970; Rees and Jones, 1972). Most of the prokaryotic genome is transcribed
(McCarthy and Bolton, 1964; Kennell, 1968; Grouse etal, 1972) and codes for
approximately 3000 informational genes, while the Drosophila and human
genomes have enough DNA to encode over 150,000 and 3 million such genes,
respectively. Since man and other eukaryotes probably do not need such a large
number of genes, explanations for the large amounts of DNA in eukaryotes have
been sought.
II. RENATURATION STUDIES OF EUKARYOTIC DNA
The first step to the solution of the apparent enigma was the demonstration
by Britten and co-workers of the presence of repetitive nucleotide sequences
within the DNA of eukaryotic organisms (Bolton et al, 1966; Waring and
Britten, 1966; Britten and Kohne, 1968). Renaturation experiments done on a
number of highly sheared animal DNA's have shown relatively constant propor-
tions of repetitive and unique sequence DNA's (Britten and Kohne, 1968;
Davidson et al, 1974). In mammals, for example, repetitive sequences usually
account for 30-40% of the genome, and unique sequences for the remaining
60-70%. Modeling after the prokaryotic system of gene regulation, transcription
and translation, it was assumed that structural gene sequences would be
represented in the unique copy DNA, whereas repetitive sequences were sus-
pected of having regulatory functions (Britten and Davidson, 1969; Georgiev,
1969). Even under this assumption, however, the question remained as to the
number of informational gene sequences present in the 60-70% of the genome
consisting of unique copy DNA.
Detailed characterization of the arrangement of repetitive and nonrepetitive
sequences was carried out in the genome of sea urchin and Xenopus (Davidson
et al, 1973; Graham et al, 1974). The techniques employed included analyses
of hydroxyapatite binding as a function of fragment length after low Cot
incubation with carrier DNA's, measurement of repetitive sequence length by
isolation of single-strand-specific, nuclease-resistant duplex on agarose gel
column, and direct measurement with electron microscopy. The results of these
studies revealed that unique copy DNA averaging 1000 nucleotides alternate
with repetitive sequences averaging 200-400 nucleotides in about 50% of the
genome. Two other patterns were also seen: long period interspersion of unique
copy DNA several thousands of nucleotides in length with a few percent of
repetitive sequences in about 40% of the genome, and clustered highly repetitive
sequences in 5-10% of the total DNA. Similar patterns have since been reported
in other eukaryotes (Firtel and Kindle, 1975; Angerer et al, 1975). The only
1. Organization and Function of the Human Genome 3
organism which has been shown to have a large digression from the above
pattern is Drosophila melanogaster (Manning et al, 1975). In these animals,
although the highly repetitive DNA sequences show a similar uninterrupted
pattern, the remainder of the genome consists of interspersed unique and
repetitive sequences covering a wide range with repetitive regions ranging from
500 to 13,000 base pairs averaging 5600, and unique copy stretches ranging
from 2500 to 40,000 base pairs and averaging 13,000 nucleotides.
In man, Schmid and Deininger (1975) have recently reported a pattern of
interspersed repetitive, single copy and inverted repeats in 50% of the genome.
The average repetitive and nonrepetitive segments were thought to be 400 and
2000 nucleotides, respectively, although measurement by either electron mi-
croscopy or isolation of single-strand, nuclease-resistant duplexes was not carried
out.
Evidence supporting the idea that unique copy sequences contiguous to
repetitive DNA represent structural genes has been presented by Davidson et al.
(1975) in the sea urchin genome. These authors have shown that 80-100% of
the mRNA molecules present in sea urchin embryos are transcribed from single
copy DNA sequences adjacent to interspersed repetitive sequences in the
genome. However, single copy DNA finely interspersed with short repetitive
sequences represents 40% of the total genome. As will be described in the
following section, the implication that these large amounts of DNA all represent
structural genes contradicts other estimates of gene number, which conclude
only 1-6% of the eukaryotic genome represents informational genes.
III. GENE NUMBER AND GENOME TRANSCRIPTION
Several lines of evidence exist in favor of the tenet that only a small
percentage of the eukaryotic genome represents structural genes. Ohta and
Kimura (1971) postulated that less than 6% of the mammalian genome repre-
sents structural genes based on mutation rate and the resultant genetic load. In
the giant salivary chromosomes of Drosophila, Judd and co-workers (Judd et ai,
1972; Judd and Young, 1974) observed that each chromomere represents one
functional genetic unit, putting the total number of informational genes in this
species at about 5000. In agreement with this view, it has been found that
cytoplasmic polysomal RNA's from a large number of eukaryotes, including
Drosophila and man, transcribe from approximately 2% of the genome (Green-
berg and Perry, 1971; Galau et αϊ, 1974; Lewin, 1975). Moreover, Bishop et al.
(1975) have shown that in Drosophila the total number of mRNA sequences ex-
pressed within the different stages of the life cycle of the fly do not exceed the
5000 chromomeres of the polytene chromosomes.
4 Jorge Yunis, Michael Tsai, and Ann Willey
In contrast to this low amount of DNA responsible for mRNA transcription,
it is known that a large amount of the DNA of the eukaryotic genome is
transcribed but not translated. On the average, assuming asymmetrical transcrip-
tion, total cellular RNA is transcribed from at least 10-30% of the single copy
DNA of the total genome (Davidson and Hough, 1971; Gelderman et al, 1971;
Grouse et al, 1972, 1973; Grady and Campbell, 1973; Turner and Laird, 1973).
Additive experiments with RNA's from different organs showed that there is a
considerable, although not total, overlap between RNA's from different tissues.
In the case of the slime mold, it was shown that, overall, 56% of the genome is
represented by transcripts between the amoeba and midculmination stages
(Firtel, 1972).
A partial explanation for the discrepancy found between the large amount of
transcribed DNA and the small percentage of the genome believed to represent
messages can be found in recent studies on heterogeneous nuclear RNA
(HnRNA). In mammalian cells, HnRNA was found to have more than five times
the complexity of mRNA (Getz et al, 1975), and in sea urchin embryos mRNA
represents 2.7% of the genome, while 28.5% of the total single copy DNA
hybridizes to HnRNA (Galau et al, 1974; Hough et al, 1975). Since there is also
evidence that a large portion of the HnRNA of the sea urchin is composed of
interspersed nonrepetitive and repetitive sequences (Smith et al, 1974), it is
possible that a large portion of the 50% finely interspersed single copy and
repetitive DNA of the sea urchin is involved in HnRNA transcription.
HnRNA's are generally characterized by their overall rapid synthesis and
degradation, large molecular weight, DNA-like base composition, their presence
in all eukaryotes examined, and by the fact that the bulk of this class of
molecules never leaves the nucleus (Sibatani et al, 1962; Georgiev and Mantieva,
1962; Scherrer et al, 1963; for review, also see Georgiev, 1974; Darnell, 1975).
Besides the unique copy sequences of HnRNA that are known to have from 5 to
10 times the complexity of mRNA, HnRNA's also contain sequences transcribed
from DNA of different degrees of repetitiveness. Holmes and Bonner (1974a)
have suggested that in rat ascites cells, HnRNA molecules contain at least one
middle repetitive sequence covalently attached to a single copy sequence. In
these cells, HnRNA's are transcribed from approximately 12% of the genome, of
which approximately 25% comes from repetitive and 75% from single copy DNA
(Holmes and Bonner, 1974a,b). One type of repetitive sequence is characterized
by its resistance to pancreatic RNase and is thought to be a double-stranded
region that is formed by intramolecular base pairing. When denatured, the RNA
sequences from the double-stranded regions, including those from He La cells,
hybridize to DNA at a Coty of about 10 (Jelinek and Darnell, 1972; Ryskovef
2
al, 1973a). A second type of repetitive sequence found in HnRNA of He La cells
is largely (about 80%) made up of uridylic acid, is about 30 nucleotides in length
and also hybridizes at a Coty of about 10 (Molloy et al, 1972). This oligo(U)
2